The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.     

生长抑素对Hela细胞中Claudin-3和Claudin-4基因表达的调节作用

目的:探讨生长抑素对Hela细胞的生长调控作用以及对claudin-3和claudin-4基因的表达调控。方法:通过Hela细胞株培养,并以浓度为10-6、10-8、10-10和10-12 M的生长抑素(SST)作用于Hela细胞,未经药物处理的细胞设为对照组。在处理后采用流式细胞仪检测Hela细胞的凋亡。并采用实时荧光定量PCR和Western blot分别检测claudin-3和claudin-4 m RNA和蛋白质表达量。结果:SST加入Hela细胞孵育12 h小时后,10-10 M、10-8 M和10-6 M浓度的SST对Hela细胞有显著性的诱导凋亡作用。不同浓度的SST作用于Hela细胞12 h后,claudin-3和claudin-4的m RNA和蛋白表达量都出现不同水平的增加。结论:在Hela细胞中SST可以促进claudin-3和claudin-4的基因表达,从而对宫颈癌的发展和扩散有抑制作用。     

靶向白介素-1α基因沉默的Hela229稳定细胞系的构建

目的：构建针对IL-1α基因的shRNA表达载体,筛选能够抑制Hela229细胞内源性IL-1α表达的shRNA,建立无内源性IL-1d表达的Hela229稳定细胞系.方法：根据shRNA的设计原则,以IL-1 αcDNA oligo为模板设计一段21 bp核苷酸目标序列,构建成siRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制IL-1α基因的重组shRNA质粒,转染Hela229细胞,经G418筛选后获得单克隆稳定细胞株,用ELISA方法在蛋白水平上检测IL-1α基因的沉默效果.结果：经酶切鉴定和测序分析确定IL-1 α-shRNA重组质粒构建正确,ELISA筛选出能够显著抑制内源性IL-1α表达的shRNA,获得沉默内源性IL-1 α表达的单克隆稳定的Hela229细胞株.结论：靶向IL-1α基因的重组shRNA表达质粒可显著抑制Hela229细胞内源性IL-1α的表达,成功构建靶向IL-1α基因沉默的Hela229稳定细胞系.     

FAM92A1-289基因的真核表达载体构建和亚细胞定位

利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。经双酶切和核酸序列分析证实重组质粒包含有正确编码的FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。     

池蝶蚌CyPA的应激表达及对Hela细胞生长的影响

研究分析了池蝶蚌(Hyriopsis schlegelii)亲环蛋白A(Cyclophilin A, CyPA)诱导的应激反应和对Hela细胞生长的影响。采用嗜水气单胞杆菌诱导刺激池蝶蚌, 并利用Real-time PCR技术对其肝脏、性腺和血淋巴中CyPA基因mRNA的表达量进行分析, 应激实验表明, 在嗜水气单胞杆菌刺激后血淋巴、肝脏和性腺中的CyPA在诱导4h出现一个表达峰, 8h后开始下降, 说明池蝶蚌HsCyPA基因参与免疫防御应答反应; 利用pET-32a(+)表达载体, 根据HsCyPA基因cDNA的序列, 构建了含有完整开放阅读框(ORF)的表达载体并在大肠杆菌BL21(DE3)中进行原核表达, 优化诱导条件后, 通过亲和层析获得了含量较高的上清可溶性蛋白; 采用MTT法, 将原核表达的重组CyPA蛋白稀释到相应(50、100、200、400和800 ng/mL)浓度, 刺激Hela细胞系后发现, 重组池蝶蚌CyPA蛋白能促进Hela细胞生长, 刺激浓度为100 ng/mL时, 达到最大增殖率18.5%。结果初步表明, 池蝶蚌CyPA是一个既参与免疫应激又对细胞的生长发育具有影响的功能广泛的蛋白质。       

Identification of a novel binding partner of phospholipase cβ1: translin-associated factor X

Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gα(q) family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gα(q). In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gα(q) for PLCβ1 binding, and excess TRAX reverses Gα(q) activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), F?rster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gα(q) for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments.     

SIRT1在宫颈癌细胞耐药中的作用及其机制研究

摘要目的：探讨沉默交配型信息调节因子2同源蛋白1（silentmatingtypeinformationregulation2homologyl,SIRTl）在宫颈癌化疗耐药中的作用及其机制。方法：体外培养人宫颈癌Hela细胞系和宫颈癌Hela／MMC耐药细胞亚系,westernblotting检测MMC对Hela和Hela／MMC细胞内SIRTl蛋白表达的影响;MTT法检测MMC及Nicotinamide对Hela和Hela／MMC细胞增殖的影响;AnnexinV-PI试验检测Hela／MMC细胞凋亡的亡的情况;RT—PCR方法检测耐药相关蛋白P—gP的mRNA表达情况。结果：正常情况下,Hela／MMC细胞中SIRTl的表达显著高于Hela细胞（P〈0．05）,MMC处理的Hela／MMC细胞中SIRTl的表达显著高于未经MMC处理（P〈0．05）。Nicotinamide对Hela和Hela／MMC细胞具有相似的生长抑制作用,Nicotinamide可使MMC诱导的Hela／MMC细胞凋亡增加,同时降低细胞内P-gP的mRNA表达（P〈0．05）。结论：SIRTl表达下调能显著减轻Hela／MMC细胞对MMC的耐药性,其作用可能与P—gp有关。     

刺参酸性粘多糖对人宫颈癌Hela细胞株增殖的影响

目的:研究刺参酸性粘多糖(SJAMP)对人宫颈癌Hela细胞株增殖的影响,并探讨其可能的作用机制.方法:体外培养Hela细胞;采用四甲基偶氮唑蓝比色法(MTT法)测定SJAMP对Hela细胞的增殖抑制率;免疫细胞化学法检测SJAMP对Hela细胞内突变型P53、细胞周期抑制蛋白P21表达的影响.结果:SJAMP在体外抑制Hela细胞生长呈时效和量效关系;与对照组比,各SJAMP浓度组均能影响细胞周期调控因子p53、p21蛋白的表达:降低p53蛋白的表达(p＜0.05),刺激p21蛋白的表达(p＜0.05).结论:SJAMP可能通过调整细胞周期调节蛋白而在体外对宫颈癌Hela细胞起到生长抑制作用.     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

MMP-7基因真核表达载体的构建及其在HeLa细胞中的表达与鉴定

目的：构建MMP-7基因真核重组质粒,检测并鉴定MMP-7在人宫颈癌HeLa细胞中的表达。方法：提取宫颈癌组织总RNA,通过基因克隆构建MMP-7基因真核表达重组质粒pcDNA3.1（＋）/MMP-7,酶切、PCR及基因测序鉴定,用阳离子脂质体介导采用基因转染技术转染人宫颈癌Hela细胞,RT-PCR检测外源基因的表达、间接免疫荧光法检测对表达产物进行鉴定。结果：成功构建了重组表达质粒pcDNA3.1（＋）/MMP-7并转染了人宫颈癌Hela细胞,通过RT-PCR可以检测到MMP-7 mRNA在Hela细胞中的表达,经间接免疫荧光反应可检测到明显的阳性反应,而转染空载体组表达阴性。结论：构建的重组质粒pcDNA3.1（＋）/MMP-7能在Hela细胞中表达,为该蛋白在人子宫癌后续的功能研究奠定了基础。     

Interferon-β Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.     

Ad5-knob蛋白在大肠杆菌中可溶性表达及其活性检测

采用PCR技术, 从AdEasy 1质粒DNA中获得knob全长序列, 克隆入pGEM T vector中。DNA测序鉴定后, 构建pQE 30/knob重组表达载体, 转化大肠杆菌M15 pREP4),IPTG诱导表达腺病毒5 型纤维蛋白的knob功能域(Ad5 knob), SDS PAGE分析,表达产物主要以可溶性蛋白的形式存在于细菌裂解液上清之中。经Ni2 NTA亲和层析一步分离纯化后,洗脱产物中Ad5 knob蛋白纯度超过95%。N 端氨基酸测序证实了纯化产物为Ad5 knob蛋白,细胞受体结合实验检测到所得蛋白能够与Hela细胞上的相应受体特异性结合。上述结果表明在大肠杆菌中已经高效表达了可溶性Ad5 knob蛋白,一步即纯化该蛋白,并具有良好的生物活性,为其下阶段的深入研究提供了重要的实验材料。     

CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的研究转染细胞周期依赖性蛋白激酶1（cyclin．dependent kinase1,CDK1）siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响,探讨CDK1在细胞凋亡中的确切作用,揭示细胞周期与细胞凋亡协调的分子机制。方法以人宫颈癌细胞株HeLa细胞为研究对象,脂质体转染CDK1siRNA,转染后48h加紫杉醇（Tax01）（20μg／m1）刺激凋亡,Western印迹检测CDK1和抗凋亡蛋白BCL2表达,AnnexinV／PI法检测细胞的凋亡,流式细胞仪分析DNA含量检测细胞周期。结果转染CDK1 siRNA后,CDK1蛋白的表达下降,细胞周期G2／M期比例增加,细胞凋亡率与对照相比没有明显升高。只加Taxol刺激12h后细胞凋亡率增加并伴有S期和G2／M期比例增加。转染CDKlsiRNA后再用Taxol刺激,其细胞凋亡率没有明显改变,G2／M期阻滞效应也没有叠加。BCL2蛋白只在加Taxol刺激组表达下降,与CDK1表达减少没有相关性。结论siRNA沉默导致的CDK1表达降低只导致细胞周期G2／M期阻滞,没有引起细胞凋亡;CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响。     

Molecular Evolution of Translin Superfamily Proteins Within the Genomes of Eubacteria, Archaea and Eukaryotes

Translin and its interacting partner protein, TRAX, are members of the translin superfamily. These proteins are involved in mRNA regulation and in promoting RISC activity by removing siRNA passenger strand cleavage products, and have been proposed to play roles in DNA repair and recombination. Both homomeric translin and heteromeric translin-TRAX complex bind to ssDNA and RNA; however, the heteromeric complex is a key activator in siRNA-mediated silencing in human and drosophila. The residues critical for RNase activity of the complex reside in TRAX sequence. Both translin and TRAX are well conserved in eukaryotes. In present work, a single translin superfamily protein is detected in Chloroflexi eubacteria, in the known phyla of archaea and in some unicellular eukaryotes. The prokaryotic proteins essentially share unique sequence motifs with eukaryotic TRAX, while the proteins possessing both the unique sequences and conserved indels of TRAX or translin can be identified from protists. Intriguingly, TRAX protein in all the known genomes of extant Chloroflexi share high sequence similarity and conserved indels with the archaeal protein, suggesting occurrence of TRAX at least at the time of Chloroflexi divergence as well as evolutionary relationship between Chloroflexi and archaea. The mirror phylogeny in phylogenetic tree, constructed using diverse translin and TRAX sequences, indicates gene duplication event leading to evolution of translin in unicellular eukaryotes, prior to divergence of multicellular eukayrotes. Since Chloroflexi has been debated to be near the last universal common ancestor, the present analysis indicates that TRAX may be useful to understand the tree of life.